Article Figures & Data

Figures

Morphological analysis of the chokh phenotype. Wild‐type and mutant embryos are shown at 24 h post‐fertilization (h.p.f.), 48 h.p.f. and 6 days post‐fertilization (d.p.f.). Eyes are absent at all stages of development in chokh (chk) mutants. The overall morphology of the head and trunk is normal in mutants. At 48 h.p.f., a small lens is visible in chk mutants (arrow). fb, forebrain; hb, hindbrain; mb, midbrain.

The chokh gene encodes Rx3. (A) Comparison of the sequencing trace data from wild‐type and chk mutant rx3 complementary DNA. Sequencing reveals a T to A point mutation (arrows). (B) The predicted translation of the wild‐type and mutant rx3 open reading frames. The mutation results in a premature stop codon (indicated by an asterisk) in the homeobox (red). (C) Comparison of zebrafish (Rx3) and medaka (OlRx3) proteins. The octapeptide (blue), homeodomain (HD; red) and the otp–aristaless–rx (OAR) domain (green) are indicated. Sequence identity at the amino‐acid level is indicated for the amino‐ and carboxy‐terminal regions and the homeodomain. The position of the nonsense mutation in the homeobox of chk and the frameshift mutation of the control plasmid (pOlRx3‐fs) are indicated (asterisks). The mutation is predicted to cause a truncation of the protein.

Whole‐mount in situ hybridization analysis of marker genes in wild‐type embryos and chokh mutants. (A–J) Dorsal views. Lateral views are shown in (K) and (L), and in the insets in (A) and (B). Anterior is to the left in all panels. (A,B) Wild‐type (WT) (A) and mutant (B) pax6a expression in the forebrain and optic vesicles at 19 h.p.f. Note the anteriorly extended pax6a expression in the mutant. Arrows indicate pax6a expression in the lens placodes. pax6a expression along the anterior–posterior axis is identical in wild‐type and mutant embryos (insets in (A) and (B), respectively). (C,D) rx3 expression in the ventral forebrain and optic vesicles of wild‐type (C) and mutant (D) embryos at 14 h.p.f. Note the ectopic rx3 expression in the forebrain of mutants (D) (arrow). (E,F) rx1 is expressed in the optic vesicles of wild‐type embryos (E) and in the forebrain of mutant embryos (F) at 14 h.p.f. Expression in the forebrain of the mutant (F) coincides with the position of the evaginated optic vesicles in wild‐type embryos (E). (G,H) rx2 is expressed in wild‐type optic vesicles (G) but not in mutants (H) at 14 h.p.f. (I–L) vsx2 expression in the optic cup and ventral midbrain and hindbrain (arrows) of wild‐type (I,K) and mutant (J,L) embryos at 26 h.p.f. Midbrain and hindbrain expression is unaffected by the mutation.

Rescue of chokh mutants by the medaka rx3 locus. (A–C) Lateral views, with the anterior to the left. (D) and (F) show transverse sections at the level of the eye. (E) is a frontal view. (A,B) Medaka rx3 (Olrx3) expression at 12 h post‐fertilization in wild‐type (WT) embryos injected with the rescue plasmid (pOlRx3; A) and control plasmid (pOlRx3‐fs; B). Note the specific expression in the ventral forebrain and optic vesicles of injected embryos. (C) The same Olrx3 probe does not cross‐hybridize with uninjected control embryos. (D–F) ath5 is expressed in ganglion cells of wild‐type retinae (D) at 36 h.p.f., but not in the mutant (E) (whole‐mount, to show the absence of staining). The dark spots in (E) are due to pigmentation. Injection of pOlRx3 restores morphology and wild‐type ath5 expression (F, compare with D).